Amberg Lab ,Upstate Medical University http://www.upstate.edu/biochem/amberg/protocols/colony_pcr.html
This procedure will work for both yeast and E. coli:
Take a small colony and suspend it in 5ul of H2O in a PCR tube. Heat for 5 min at 95¡C and then spin the condensation down in a microfuge. Set up the PCR reaction as follows:
- 5ul H2O cells
- 5ul 5uM primer2
- 5ul 10XTaq Buffer
- 5ul 2mM dNTPs
- 0.5ul 10 mg/ml acetylated BSA
- 1ul Taq DNA polymerase
- 23.5 ul H2O
PCR Conditions: 94¡C x 4min. then 35 cycles of: (94¡C x 1 min then 55¡C x 1min then 72¡C x 3min) followed by 72¡C x 20 min and a 4¡C soak. (5ul run out on a mini gel should be sufficient to see product.)
- 10X TAQ Buffer:
- 0.2M TRIS pH8.3
- 15mM MgCl2
- 0.25M KCl
- 0.5% TWEEN 20
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